Our long-term goal is to study the role of protein synthesis in programmed cell death. Inhibition of protein synthesis is known to block programmed cell death and protect tumor cells from the cytotoxic action of many anti-tumor drugs. In the current proposal, we plan to investigate the role of eEf-2 kinase in regulating protein synthesis and programmed cell death. eEF-2 kinase, a ubiquitous protein kinase, whose function is to phosphorylate elongation factor-2 (eEF-2) and arrest protein synthesis (Ryazanov et al. (1988) Nature 334: 170-173). We recently cloned and sequenced eEF-2 kinase from various species and discovered that it is a member of a novel class of protein kinases that are structurally and evolutionarily unrelated to conventional eukaryotic protein kinases (Ryazanov et. al (1997) Proc. Nat. Acad. Sci. USA 94: 4884-4889). In preliminary studies, we have obtained evidence that increased eEF-2 kinase expression, which has been found in many human tumors, can confer resistance of cells to anti-cancer drugs. In addition, we found that eEF-2 kinase is strongly activated by a slight decrease in pH, a condition normally found in hypoxic solid tumors. We hypothesize that pH- dependent activation of eEF-2 kinase, which arrests protein synthesis, is a major protective mechanism of cells against programmed cell death and the cytotoxic action of anti-cancer drugs. Consequently, inhibition of eEF-2 kinase is expected to sensitize cells against apoptotic stimuli and anti-cancer drugs. Our ultimate goal is to develop specific inhibitors of eEF-2 kinase in order to sensitize solid tumors to programmed cell death. Specifically, the role of eEF-2 kinase in programmed cell death will be evaluated by construction of eEF-2 kinase knockout mice. The resistance/sensitivity of cells lacking eEF-2 kinase to anti-cancer drugs will be analyzed. Mechanistic studies will be performed to reveal the exact position of eEF-2 kinase in the network of apoptotic signal transduction cascades. We will investigate the molecular properties of eEF-2 kinase a potential drug target. In preliminary studies, we have shown that eEF-2 kinase has an unusual catalytic domain, and may recognize alpha-helical conformation in its substrate. Experiments will be performed to design and develop specific eEF-2 kinase inhibitors which can serve as pro- apoptotic agents.